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Oxidation of Malate by Isolated Plant OXIDATION OF MALATE BY ISOLATED PLANT MITOCHONDRIA by NAJAT ALI AL-SANE B.Sc., M.Sc, January 1981 A thesis submitted for the degree of Doctor of Philosophy of the University of London and for the Diploma of Imperial College Department of Botany and Plant Technology, Imperial College London SW7. -2- ABSTRACT The characteristics of malate oxidation by Jerusalem artichoke mitochondria were studied with special attention to the influence of added coenzymes. Thiamine pyrophosphate was found to increase the rate of oxygen uptake suggesting that one of the factors regulating the rate of malate oxidation was the conversion of pyruvate, which is produced as a result of malic enzyme activity, to acetyl-CoA which could subsequently remove oxaloacetate produced as a result of malate dehydrogenase activity thus displacing the equilibrium of the malate + dehydrogenase reaction. Exogenous NAD stimulated oxygen uptake in the presence of malate and had less effect when citrate was the sub- strate. Piericidin A severely inhibited the oxidation of malate in the presence of oxaloacetate. These results suggest that under such con- ditions only the piericidin A-sensitive pathway was involved in the oxidation of NADH produced from malate. The effect of n-butyl malonate on the rate of malate oxidation was also studied and results obtained showed that malate oxidation was sensitive to this inhibitor, in the absence of NAD+, while the NAD+-stimulated rate was not affected by n-butylmalonate. From these results it was concluded that malate oxidation by Jerusalem artichoke mitochondria, takes place through two pathways, one located in the matrix space (transport-dependent) and is associated with the internal NADH dehydrogenase system. And the other takes place in the outer compartment (transport-independent) and depends on added NAD . Oxi- dation through this pathway is associated with the external NADH de- hydrogenase system. -3- Endogenous pyridine nucleotide contents were determined for washed and purified Jerusalem artichoke and mung bean mitochondria. Mung bean mitochondria were found to contain twice that of Jerusalem artichoke mitochondria. Thiamine pyrophosphate contents were also determined for both types of mitochondria. Further purification of mitochondria was carried out using Percoll density gradient. Malate oxidation by purified Jerusalem artichoke mitochondria was stimulated by added NAD+. In addition Jerusalem artichoke mitochondria could reduce exogenous NAD+ when malate was the substrate, but not when citrate was the substrate. The enzymes responsible for malate oxidation were purified from isolated Jerusalem artichoke mitochondria and some of their kinetic properties were studied. Purified malic enzyme showed a requirement 2+2+ + for Mn or Mg and required NAD as a cofactor. Malate dehydrogen- ase was found to be present in a large quantity when compared with + + malic enzyme and required NAD as a cofactor. The IC^ for NAD of both enzymes were almost of the same magnitude. -4- ACKNOWLEDGEMENT First of all I should like to express my sincere gratitude to Dr.J.M. Palmer for his great help and patient encouragement through- out this investigation. My thanks are due to Miss Suzanne Cheston for typing the manu- script and to Mrs. Jill Farmer for her help during this study. I am in debt to my family for their financial and moral support. I would like to extend my thanks to the University of Kuwait for the financial support. -5- CONTENTS ABSTRACT 2 ACKNOWLEDGEMENTS 4 ABBREVIATIONS 8 SYMBOLS 9 LIST OF FIGURES 10 LIST OF TABLES 12 INTRODUCTION 1. General Characteristics of Plant Mitochondria 13 a. Cyanide-resistant pathway 13 b. NADH-dehydrogenase Systems 16 c. Malate oxidation 18 2. Location of enzymes responsible for Malate Oxidation ... 19 a. Dual location 20 b. Transmembrane transhydrogenase 22 3. Purification of Mitochondria 24 4. Aim of the Study 25 MATERIALS AND METHODS 1. Chemicals 27 2. Media Used 27 3. Isolation of Mitochondria 28 a. Jerusalem artichoke mitochondria 28 b. Potato mitochondria 29 c. Mung bean mitochondria 29 d. Rat liver mitochondria 30 4. Assays and analytical procedures 30 a. Oxygen consumption 30 -6- b. Determination of ADP/O and respiratory control ratios 31 c. Determination of ADP and AMP concentration 31 d. Determination of protein contents 32 e. Activation of succinate dehydrogenase 32 f. Succinate dehydrogenase assay 32 g. Succinate cytochrome c reductase assay 33 h. Reduction of exogenous NAD+ 33 5. Purification of Mitochondria 34 6. Determination of endogenous NAD+ contents 35 7. Determination of endogenous TPP contents 36 + 8. Purification of NAD -linked malic enzyme 36 a. Isolation of mitochondria 36 b. Sonication 37 c. Ammonium Sulphate fractionation 37 d. Gel filtration 37 e. DEAE-Sephadex 38 f. Assay of enzyme activity 38 9. Purification of NAD+-linked malate dehydrogenase 39 a. Ammonium sulphate fractionation 39 b. Sephadex S-200 40 c. Sephadex G-25 40 d. DEAE-Sephadex 40 e. Assay of malate dehydrogenase 41 RESULTS 1. General characteristics of Jerusalem artichoke mitochondria 42 2. Pyruvate oxidation 46 3. Malate oxidation 48 -7- a. Effect of thiamine pyrophosphate 50 + b. Effect of exogenous NAD 53 c. Effect of piericidin A 56 d. Effect of butyl malonate 58 e. Effect of added oxaloacetate 64 4. Purification of Mitochondria 68 a. Integrity of mitochondrial preparations 73 b. Malate oxidation 80 c. Endogenous NAD+ and TPP contents 83 d. Permeability to NAD+ 85 + e. Reduction of exogenous NAD 87 5. Purification of enzymes responsible for malate oxidation 91 a. Purification of NAD+-malic enzyme 91 b. Purification of NAD+-malate dehydrogenase V.. 99 DISCUSSION 108 1. Effect of Inhibitors 110 a. Effect of piericidin A 110 b. Effect of n-butyl malonate 112 c. Effect of oxaloacetate 113 2. Effect of exogenous NAD+ 115 3. Reduction of exogenous NAD+ 117 4. Purification of mitochondria 119 5. Malate oxidizing enzymes 121 REFERENCES 125 APPENDIX Palmer, J.M., Cowley, R.C. & Al-Sane, N.A. (1978). The inhibition of malate oxidation by oxaloacetate in Jerusalem artichoke mitochondria. 'Plant Mitochondria' (Ducet, G. & Lance, C. eds.) Elsevier, Amsterdam. -8- ABBREVIATIONS ADP Adenosine-5'-diphosphate AMP Adenosine-5'-monophosphate ATP Ad eno s ine-5'-tr ipho sphat e BM n-butylmalonate BSA Bovine serum albumin (fraction V) DCIP 2,6-Dichlorophenol-indophenol DTT Dithiothretol EDTA Ethylenediamine tetraacetic acid MOPS 3-(N-morpholino) propane sulphonic acid NAD+ Nicotinamide-adenine dinucleotide (oxidized) NADH Nicotinamide-adenine dinucleotide (reduced) OAA Oxaloacetate P/A Piericidin A PEP Phosphoenol pyruvate PK Pyruvate kinase PMS Phenazine methosulphate S.E. Standard error TES N-tris(hydroxymethyl)-methyl-2-aminoethane sulphonic acid TPP Thiamine pyrophosphate Triton X 100 Octylphenoxypolyethoxyethanol SYMBOLS maximum velocity of reaction Michaelis constant, the concentration of substrate permitting half V ° max -2 acceleration due to gravity (981 cm.s ) -10- LIST OF FIGURES page 1 Malate oxidation by Jerusalem artichoke and rat liver mitochondria 45 2 Pyruvate oxidation by Jerusalem artichoke mito- chondria 47 3 Malate oxidation by Jerusalem artichoke mito- 49 chondria 4 Effect of cofactors on malate oxidation 51 + 5 Effect of exogenous NAD and TPP on malate oxi- dation 52 6 Effect of piericidin A on malate oxidation 57 7 Effect of n-butylmalonate on malate oxidation 59 8 Effect of n-butylmalonate on NAD+-stimulated rate 62 9 Effect of oxaloacetate on citrate oxidation 66 10 Effect of oxaloacetate on malate oxidation 67 11 Development of Percoll density gradient 71 12 Distribution of Jerusalem artichoke mitochondria on 20% Percoll density gradient 72 13 Distribution of Jerusalem artichoke mitochondria on 18% Percoll density gradient 74 14 Distribution of mung bean mitochondria on Percoll density gradient 75 15 Distribution of potato mitochondria on Percoll density gradient 76 16 Exogenous NAD+ reduction by Jerusalem artichoke mitochondria 88 17 Purification of malic enzyme 93 18 Activity of malic enzyme as a function of NAD+ concentration in the presence of 10 mM Malate 95 19 Activity of malic enzyme as a function of NAD+ concentration in the presence of 50 mM Malate 96 -11- page 20 Linweaver-Burk plot of reaction of velocity of malic enzyme as a function of NAD concentrations in the presence of MnCl^ 97 21 Linweaver-Burk plot of reaction velocity of malic enzyme as a function of NAD concentrations in the presence of MgCl^ 98 22 The rate of malate dehydrogenase activity as a function of enzyme concentration 102 23 Malate dehydrogenase activity as a function of malate concentration 103 24 Mal^te dehydrogenase activity as a function of NAD concentration 105 25 Linweaver-Burk glot of reaction velocity as a function of NAD concentration 106 26 Linweaver-Burk plot of reaction velocity as a function of malate concentration 107 -12- LIST OF TABLES page 1 Rate of oxidation of different substrates 43 2 Stimulation of oxygen uptake by exogenous NAD+ 55 3 Effect of BM on state 3 and NAD+-stimulated rates of malate oxidation 61 4 Effect of BM on enzyme activity 63 5 Cytochrome c reduction by washed and purified mitochondria 77 6 NADH oxidation by washed and purified mito- chondria 79 7 Effect of NAD+ and TPP on malate oxidation by washed and purified Jerusalem artichoke mito- chondria 81 8 Effect of NAD+ and TPP on malate oxidation by washed and purified mung bean mitochondria 82 -f 9 Endogenous NAD and TPP contents 84 10 NAD+ uptake by Jerusalem artichoke mitochondria 86 + 11 Reduction of exogenous NAD by Jerusalem
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